Achromosomal dynamic active systems

Abstract

The invention provides isolated achromosomal dynamic active systems (ADAS), including highly active ADAS. These ADAS provided by the invention can be obtained by a variety of means. Various associated methods of making and using these ADAS are provided.

What is claimed is: 
     
         1 . A method for manufacturing a composition comprising a plurality of ADAS, the composition being substantially free of viable bacterial cells, the method comprising:
 (a) making, providing, or obtaining a plurality of parent bacteria having a reduction in the level or activity of a cell division topological specificity factor;   (b) exposing the parent bacteria to conditions allowing the formation of minicells; and   (c) separating the minicells from the parent bacteria, thereby producing a composition comprising a plurality of ADAS that is substantially free of viable bacterial cells.   
     
     
         2 . The method of  claim 1 , wherein the cell division topological specificity factor is a polypeptide having an amino acid sequence with at least 90% identity to SEQ ID NO: 1. 
     
     
         3 . The method of  claim 1  or  2 , wherein the cell division topological specificity factor is a polypeptide having an amino acid sequence with at least 95% identity to SEQ ID NO: 1. 
     
     
         4 . The method of any one of  claims 1 - 3 , wherein the cell division topological specificity factor is a minE polypeptide. 
     
     
         5 . The method of  claim 4 , wherein the parent bacteria are  E. coli  and the minE polypeptide is  E. coli  minE. 
     
     
         6 . The method of  claim 4 , wherein the parent bacteria are  Salmonella typhimurium  and the minE polypeptide is  S. typhimurium  minE. 
     
     
         7 . The method of any one of  claims 1 - 6 , wherein the parent bacteria have a reduction in the level or activity of a Z-ring inhibition protein. 
     
     
         8 . The method of  claim 7 , wherein the Z-ring inhibition protein is a polypeptide having an amino acid sequence with at least 90% identity to SEQ ID NO: 2. 
     
     
         9 . The method of any of  claim 7  or  8 , wherein the Z-ring inhibition protein is a polypeptide having an amino acid sequence with at least 90% identity to SEQ ID NO: 3. 
     
     
         10 . The method of  claim 8 , wherein the Z-ring inhibition protein is a minC polypeptide. 
     
     
         11 . The method of  claim 9 , wherein the Z-ring inhibition protein is a minD polypeptide. 
     
     
         12 . The method of any one of  claims 1 - 11 , wherein the ADAS have a reduction in expression of at least two Z-ring inhibition proteins. 
     
     
         13 . The method of  claim 12 , wherein the ADAS have a reduction in expression of a minC polypeptide and a minD polypeptide. 
     
     
         14 . The method of  claim 13 , wherein the ADAS have a reduction in expression of a minC polypeptide, a minD polypeptide, and a minE polypeptide. 
     
     
         15 . The method of  claim 1 , wherein the cell division topological specificity factor is a polypeptide having an amino acid sequence with at least 90% identity to SEQ ID NO: 4. 
     
     
         16 . The method of  claim 15 , wherein the cell division topological specificity factor is a polypeptide having an amino acid sequence with at least 95% identity to SEQ ID NO: 4. 
     
     
         17 . The method of any one of  claims 1 , 15, and 16, wherein the cell division topological specificity factor is a DivIVA polypeptide. 
     
     
         18 . The method of  claim 17 , wherein the parent bacteria are  Bacillus subtilis  and the cell division topological specificity factor is  B. subtilis  DivIVA. 
     
     
         19 . The method of any one of  claims 1 - 18 , wherein the reduction in the level or activity is caused by a loss-of-function mutation. 
     
     
         20 . The method of  claim 19 , wherein the loss-of-function mutation is a deletion of the minCDE operon or a deletion of DiVIVA. 
     
     
         21 . The method of any of  claims 1 - 20 , wherein the ADAS have an initial ATP concentration of at least 1 mM, 1.2 nM, 1.3 nM, 1.4 mM, 1.5 mM, 1.6 mM, 2 mM, 2.5 mM, 3 mM, 4 mM, 5 mM, 10 mM, 20 mM, 30 mM, or 50 mM. 
     
     
         22 . The method of  claim 21 , wherein the parent bacteria are  Escherichia, Acinetobacter, Agrobacterium, Anabaena, Aquifex, Azoarcus, Azotobacter, Bordetella, Bradyrhizobium, Brucella, Buchnera, Burkholderia, Candidatus, Chromobacterium, Crocosphaera, Dechloromonas, Desulfitobacterium, Desulfotalea, Erwinia, Francisella, Fusobacterium, Gloeobacter, Gluconobacter, Helicobacter, Legionella, Magnetospirillum, Mesorhizobium, Methylococcus, Neisseria, Nitrosomonas, Nostoc, Photobacterium, Photorhabdus, Polaromonas, Prochlorococcus, Pseudomonas, Psychrobacter, Ralstonia, Rubrivivax, Salmonella, Shewanella, Shigella, Sinorhizobium, Synechococcus, Synechocystis, Thermosynechococcus, Thermotoga, Thermus, Thiobacillus, Trichodesmium, Vibrio, Wigglesworthia, Wolinella, Xanthomonas, Xylella, Yersinia, Bacillus, Clostridium, Deinococcus, Exiguobacterium, Geobacillus, Lactobacillus, Lactobacillus, Moorella, Oceanobacillus, Symbiobacterium , or  Thermoanaerobacter  bacteria and the cell division topological specificity factor is the endogenous minE or DivIVA of the parent bacteria. 
     
     
         23 . The method of any of  claims 1 - 22 , wherein the composition of step (c) comprises less than 100 colony-forming units (CFU/mL) of viable bacterial cells. 
     
     
         24 . The method of  claim 23 , wherein the composition of step (c) comprises less than 10 CFU/mL, less than 1 CFU/mL, or less than 0.1 CFU/mL of viable bacterial cells. 
     
     
         25 . The method of any of  claims 1 - 24 , wherein the ADAS comprise a cargo. 
     
     
         26 . The method of any of  claims 1 - 25 , wherein the composition is formulated for delivery to an animal. 
     
     
         27 . The method of any of  claims 1 - 25 , wherein the composition is formulated for delivery to a plant. 
     
     
         28 . The method of any of  claims 1 - 25 , wherein the composition is formulated for delivery to an insect. 
     
     
         29 . The method of any one of  claims 1 - 28 , wherein the composition is formulated as a liquid, a solid, an aerosol, a paste, a gel, or a gas composition. 
     
     
         30 . A composition comprising a plurality of highly active achromosomal dynamic active systems (ADAS), wherein the ADAS have an initial ATP concentration of at least 1 mM and wherein the composition is substantially free of viable bacterial cells. 
     
     
         31 . The composition of  claim 30 , wherein the ADAS have an initial ATP concentration of at least 1.2 nM, 1.3 nM, 1.4 mM, 1.5 mM, 1.6 mM, 2 mM, or 2.5 mM. 
     
     
         32 . A composition comprising a plurality of highly active ADAS, wherein the ADAS have an initial ATP concentration of at least 3 mM and wherein the composition is substantially free of viable bacterial cells. 
     
     
         33 . The composition of  claim 32 , wherein the ADAS have an initial ATP concentration of at least 4 mM, 5 mM, 10 mM, 20 mM, 30 mM, or 50 mM. 
     
     
         34 . The composition of any one of  claims 30 - 33 , wherein the ATP concentration of the ADAS is increased by at least 50%, at least 60%, at least 75%, at least 100%, at least 150%, or at least 200% following incubation at 37° C. for 12 hours. 
     
     
         35 . The composition of any one of  claims 30 - 34 , wherein the ADAS are derived from parent bacteria having a reduction in a level or activity of a cell division topological specificity factor. 
     
     
         36 . A composition comprising a plurality of ADAS, wherein the ADAS do not comprise a cell division topological specificity factor and wherein the composition is substantially free of viable bacterial cells. 
     
     
         37 . A composition comprising a plurality of ADAS, the composition being substantially free of viable bacterial cells, and being produced by a process comprising:
 (a) making, providing, or obtaining a plurality of parent bacteria having a reduction in the level or activity of a cell division topological specificity factor;   (b) exposing the parent bacteria to conditions allowing the formation of minicells; and   (c) separating the minicells from the parent bacteria, thereby producing a composition that is substantially free of viable bacterial cells.   
     
     
         38 . The composition of any one of  claims 30 - 37 , wherein the cell division topological specificity factor is a polypeptide having an amino acid sequence with at least 90% identity to SEQ ID NO: 1. 
     
     
         39 . The composition of  claim 38 , wherein the cell division topological specificity factor is a polypeptide having an amino acid sequence with at least 95% identity to SEQ ID NO: 1. 
     
     
         40 . The composition of any one of  claims 30 - 37 , wherein the cell division topological specificity factor is a minE polypeptide. 
     
     
         41 . The composition of  claim 40 , wherein the parent bacteria is  E. coli  and the minE polypeptide is  E. coli  minE. 
     
     
         42 . The composition of  claim 42 , wherein the parent bacteria is  Salmonella typhimurium  and the minE polypeptide is  S. typhimurium  minE. 
     
     
         43 . The composition of any of  claims 37 - 42 , wherein the parent bacteria are  Escherichia, Acinetobacter, Agrobacterium, Anabaena, Aquifex, Azoarcus, Azotobacter, Bordetella, Bradyrhizobium, Brucella, Buchnera, Burkholderia, Candidatus, Chromobacterium, Crocosphaera, Dechloromonas, Desulfitobacterium, Desulfotalea, Erwinia, Francisella, Fusobacterium, Gloeobacter, Gluconobacter, Helicobacter, Legionella, Magnetospirillum, Mesorhizobium, Methylococcus, Neisseria, Nitrosomonas, Nostoc, Photobacterium, Photorhabdus, Polaromonas, Prochlorococcus, Pseudomonas, Psychrobacter, Ralstonia, Rubrivivax, Salmonella, Shewanella, Shigella, Sinorhizobium, Synechococcus, Synechocystis, Thermosynechococcus, Thermotoga, Thermus, Thiobacillus, Trichodesmium, Vibrio, Wigglesworthia, Wolinella, Xanthomonas, Xylella, Yersinia, Bacillus, Clostridium, Deinococcus, Exiguobacterium, Geobacillus, Lactobacillus, Lactobacillus, Moorella, Oceanobacillus , Symbiobacterium, or  Thermoanaerobacter  bacteria and the cell division topological specificity factor is the endogenous minE or DivIVA of the parent bacteria. 
     
     
         44 . The composition of any one of  claims 30 - 43 , wherein the ADAS have a reduction in a level of a Z-ring inhibition protein. 
     
     
         45 . The composition of  claim 44 , wherein the Z-ring inhibition protein is a polypeptide having an amino acid sequence with at least 90% identity to SEQ ID NO: 2. 
     
     
         46 . The composition of  claim 44 , wherein the Z-ring inhibition protein is a polypeptide having an amino acid sequence with at least 90% identity to SEQ ID NO: 3. 
     
     
         47 . The composition of  claim 44 , wherein the Z-ring inhibition protein is a minC polypeptide. 
     
     
         48 . The composition of  claim 44 , wherein the Z-ring inhibition protein is a minD polypeptide. 
     
     
         49 . The composition of any one of  claims 44 - 48 , wherein the ADAS have a reduction in expression of at least two Z-ring inhibition proteins. 
     
     
         50 . The composition of  claim 49 , wherein the ADAS have a reduction in expression of a minC polypeptide and a minD polypeptide. 
     
     
         51 . The composition of  claim 50 , wherein the ADAS have a reduction in expression of a minC polypeptide, a minD polypeptide, and a minE polypeptide. 
     
     
         52 . The composition of any one of  claims 35 - 51 , wherein the cell division topological specificity factor is a polypeptide having an amino acid sequence with at least 90% identity to SEQ ID NO: 4. 
     
     
         53 . The composition of  claim 52 , wherein the cell division topological specificity factor is a polypeptide having an amino acid sequence with at least 95% identity to SEQ ID NO: 4. 
     
     
         54 . The composition of  claim 52  or  53 , wherein the cell division topological specificity factor is a DivIVA polypeptide. 
     
     
         55 . The composition of  claim 53  or  54 , wherein the parent bacteria are  Bacillus subtilis  and the cell division topological specificity factor is  B. subtilis  DivIVA. 
     
     
         56 . The composition of any one of  claims 35 - 51 , wherein the reduction in the level or activity is caused by a loss-of-function mutation. 
     
     
         57 . The composition of  claim 56 , wherein the loss-of-function mutation is a gene deletion. 
     
     
         58 . The composition of  claim 56  or  57 , wherein the loss-of-function mutation is an inducible loss-of-function mutation and wherein loss of function is induced by exposing the parent cell to an inducing condition. 
     
     
         59 . The composition of  claim 58 , wherein the inducible loss-of-function mutation is a temperature-sensitive mutation and wherein the inducing condition is a temperature condition. 
     
     
         60 . The composition of  claim 59 , wherein the parent cell has a deletion of the minCDE operon. 
     
     
         61 . The composition of any one of  claims 1 - 60 , wherein the ADAS comprise a functional transcription system and a functional translation system. 
     
     
         62 . The composition of  claim 61 , wherein the ADAS produce a heterologous protein. 
     
     
         63 . The composition of  claim 62 , wherein the ADAS comprise a plasmid, the plasmid comprising an inducible promoter and a nucleotide sequence encoding the heterologous protein, and wherein contacting the ADAS with an inducer of the inducible promoter under appropriate conditions results in production of the heterologous protein. 
     
     
         64 . The composition of  claim 63 , wherein production of the heterologous protein is increased by at least 1.6-fold in an ADAS that has been contacted with the inducer relative to an ADAS that has not been contacted with the inducer. 
     
     
         65 . The composition of  claim 64 , wherein the rate of production of the heterologous protein reaches a target level within 3 hours of the contacting of the ADAS with the inducer. 
     
     
         66 . The composition of any one of  claims 64 - 65 , wherein the heterologous protein is produced at a rate of at least 0.1 femtograms per hour per ADAS. 
     
     
         67 . The composition of any one of  claims 62 - 66 , wherein the heterologous protein is produced for a duration of at least 8 hours. 
     
     
         68 . The composition of any one of  claims 1 - 67 , wherein the composition comprises less than 100 colony-forming units (CFU/mL) of viable bacterial cells. 
     
     
         69 . The composition of  claim 68 , wherein the composition comprises less than 10 CFU/mL, less than 1 CFU/mL, or less than 0.1 CFU/mL of viable bacterial cells. 
     
     
         70 . The composition of any one of  claims 1 - 69 , wherein the ADAS comprise a cargo. 
     
     
         71 . The composition of  claim 70 , wherein the cargo is a nucleic acid, a plasmid, a polypeptide, a protein, an enzyme, an amino acid, a small molecule, a gene editing system, a hormone, an immune modulator, a carbohydrate, a lipid, an organic particle, an inorganic particle, or a ribonucleoprotein complex (RNP). 
     
     
         72 . The composition of  claim 71 , wherein the cargo is encapsulated by the ADAS. 
     
     
         73 . The composition of  claim 71 , wherein the cargo is attached to the surface of the ADAS. 
     
     
         74 . The composition of  claim 71 , wherein the nucleic acid is a DNA, an RNA, or a plasmid. 
     
     
         75 . The composition of  claim 71  or  74 , wherein the nucleic acid encodes a protein. 
     
     
         76 . The composition of  claim 71 , wherein the enzyme alters a substrate to produce a target product. 
     
     
         77 . The composition of  claim 76 , wherein the substrate is present in the ADAS and wherein the target product is produced in the ADAS. 
     
     
         78 . The composition of  claim 76 , wherein the substrate is present in a target cell or environment to which the ADAS is delivered. 
     
     
         79 . The composition of any one of  claims 1 - 78 , wherein the ADAS comprises a heterologous bacterial secretion system. 
     
     
         80 . The composition of  claim 79 , wherein the heterologous bacterial secretion system is a type 3 secretion system (T3SS). 
     
     
         81 . The composition of any one of  claims 79 - 80 , wherein the cargo comprises a moiety that directs export by the bacterial secretion system. 
     
     
         82 . The composition of any one of  claims 1 - 81 , wherein the ADAS comprises a targeting moiety. 
     
     
         83 . The composition of  claim 82 , wherein the targeting moiety is a nanobody, a carbohydrate binding protein, or a tumor-targeting peptide. 
     
     
         84 . The composition of any one of  claims 1 - 83 , wherein the ADAS have a reduced protease level or activity relative to an ADAS produced from a wild-type parent bacterium. 
     
     
         85 . The composition of  claim 84 , wherein the ADAS is produced from a parent bacterium that has been modified to reduce or eliminate expression of at least one protease. 
     
     
         86 . The composition of any one of  claims 1 - 85 , wherein the ADAS have a reduced RNase level or activity relative to an ADAS produced from a wild-type patent bacterium. 
     
     
         87 . The composition of  claim 86 , wherein the ADAS is produced from a parent bacterium that has been modified to reduce or eliminate expression of at least one RNase. 
     
     
         88 . The composition of  claim 86  or  87 , wherein the RNase is an endoribonuclease or an exoribonuclease. 
     
     
         89 . The composition of any one of  claims 1 - 88 , wherein the ADAS has been modified to have reduced lipopolysaccharide (LPS). 
     
     
         90 . The composition of  claim 60 , wherein the ADAS is produced from parent bacteria that have been modified to have reduced LPS. 
     
     
         91 . The composition of  claim 89 , wherein the modification is a mutation in Lipid A biosynthesis myristoyltransferase (msbB). 
     
     
         92 . The composition of any one of  claims 1 - 91 , wherein the ADAS is derived from parent bacteria that are a mammalian pathogen or a mammalian commensal bacterium. 
     
     
         93 . The composition of  claim 92 , wherein the mammalian commensal bacterium is  Staphylococcus, Bifidobacterium, Micrococcus, Lactobacillus , or  Actinomyces  species or the mammalian pathogenic bacterium is enterohemorrhagic  Escherichia coli  (EHEC),  Salmonella typhimurium, Shigella flexneri, Yersinia enterolitica , or  Helicobacter pylori.    
     
     
         94 . The composition of any one of  claims 1 - 91 , wherein the ADAS is derived from parent bacteria that a plant pathogen or a plant commensal bacterium. 
     
     
         95 . The composition of  claim 94 , wherein the plant commensal bacterium is  Bacillus subtilis  or  Psuedomonas putida  or the plant pathogenic bacterium is a  Xanthomonas  species or  Pseudomonas syringae.    
     
     
         96 . The composition of any one of  claims 1 - 95 , wherein the ADAS is derived from auxotrophic parent bacteria. 
     
     
         97 . The composition of any one of  claims 1 - 96 , wherein the ADAS are lyophilized and reconstituted, and wherein the reconstituted ADAS have an ATP concentration that is at least 95% of the ATP concentration of an ADAS that has not been lyophilized. 
     
     
         98 . The composition of  claim 97 , wherein the reconstituted ADAS have an ATP concentration that is at least equal to the ATP concentration of an ADAS that has not been lyophilized. 
     
     
         99 . The composition of any one of  claims 1 - 98 , wherein the composition is formulated for delivery to an animal. 
     
     
         100 . The composition of  claim 99 , wherein the composition is formulated for intraperitoneal, intravenous, intramuscular, oral, topical, aerosolized, or nebulized administration. 
     
     
         101 . The composition of any one of  claims 1 - 98 , wherein the composition is formulated for delivery to a plant. 
     
     
         102 . The composition of any one of  claims 1 - 97 , wherein the composition is formulated for delivery to an insect. 
     
     
         103 . The composition of any one of  claims 99 - 102 , wherein the composition is formulated as a liquid, a solid, an aerosol, a paste, a gel, or a gas composition. 
     
     
         104 . A method for delivering a highly active ADAS to a target cell, the method comprising:
 (a) providing a composition comprising a plurality of highly active ADAS, wherein the ADAS have an initial ATP concentration of at least 1.25 mM and wherein the composition is substantially free of viable bacterial cells; and   (b) contacting the target cell with the composition of step (a).   
     
     
         105 . A method for delivering an ADAS to a target cell, the method comprising:
 (a) providing a composition comprising a plurality of ADAS, wherein the ADAS are derived from parent bacteria having a reduction in the level or activity of a cell division topological specificity factor and wherein the composition is substantially free of viable bacterial cells; and   (b) contacting the target cell with the composition of step (a).   
     
     
         106 . The method of  claim 104  or  105 , wherein the target cell is an animal cell. 
     
     
         107 . The method of  claim 104  or  105 , wherein the target cell is a plant cell. 
     
     
         108 . The method of  claim 104  or  105 , wherein the target cell is an insect cell. 
     
     
         109 . A method for delivering a cargo to a target cell, the method comprising:
 (a) providing a composition comprising a plurality of highly active achromosomal dynamic active systems (ADAS), wherein the ADAS have an initial ATP concentration of at least 1.25 mM, the ADAS comprise a cargo, and the composition is substantially free of viable bacterial cells; and   (b) contacting the target cell with the composition of step (a).   
     
     
         110 . A method for delivering a cargo to a target cell, the method comprising:
 (a) providing a composition comprising a plurality of ADAS, wherein the ADAS are derived from parent bacteria having a reduction in the level or activity of a cell division topological specificity factor, the ADAS comprise a cargo, and the composition is substantially free of viable bacterial cells; and   (b) contacting the target cell with the composition of step (a).   
     
     
         111 . The method of  claim 109  or  110 , wherein the target cell is an animal cell. 
     
     
         112 . The method of  claim 109  or  110 , wherein the target cell is a plant cell. 
     
     
         113 . The method of  claim 109  or  110 , wherein the target cell is an insect cell. 
     
     
         114 . A method of modulating a state of an animal cell, the method comprising:
 (a) providing a composition comprising a plurality of highly active achromosomal dynamic active systems (ADAS), wherein the ADAS have an initial ATP concentration of at least 1.25 mM and wherein the composition is substantially free of viable bacterial cells; and   (b) contacting the animal cell with the composition of step (a), whereby a state of the animal cell is modulated.   
     
     
         115 . A method of modulating a state of a plant cell, the method comprising:
 (a) providing a composition comprising a plurality of highly active achromosomal dynamic active systems (ADAS), wherein the ADAS have an initial ATP concentration of at least 1.25 mM and wherein the composition is substantially free of viable bacterial cells; and   (b) contacting the plant cell with the composition of step (a), whereby a state of the plant cell is modulated.   
     
     
         116 . A method of modulating a state of an insect cell, the method comprising:
 (a) providing a composition comprising a plurality of highly active achromosomal dynamic active systems (ADAS), wherein the ADAS have an initial ATP concentration of at least 1.25 mM and wherein the composition is substantially free of viable bacterial cells; and   (b) contacting the insect cell with the composition of step (a), whereby a state of the insect cell is modulated.   
     
     
         117 . A method of modulating a state of an animal cell, the method comprising:
 (a) providing a composition comprising a plurality of ADAS, wherein the ADAS are derived from parent bacteria having a reduction in the level or activity of a cell division topological specificity factor and wherein the composition is substantially free of viable bacterial cells; and   (b) contacting the animal cell with the composition of step (a), whereby a state of the animal cell is modulated.   
     
     
         118 . A method of modulating a state of a plant cell, the method comprising:
 (a) providing a composition comprising a plurality of ADAS, wherein the ADAS are derived from parent bacteria having a reduction in the level or activity of a cell division topological specificity factor and wherein the composition is substantially free of viable bacterial cells; and   (b) contacting the plant cell with the composition of step (a), whereby a state of the plant cell is modulated.   
     
     
         119 . A method of modulating a state of an insect cell, the method comprising:
 (a) providing a composition comprising a plurality of ADAS, wherein the ADAS are derived from parent bacteria having a reduction in the level or activity of a cell division topological specificity factor and wherein the composition is substantially free of viable bacterial cells; and   (b) contacting the insect cell with the composition of step (a), whereby a state of the insect cell is modulated.   
     
     
         120 . A method of treating an animal in need thereof, the method comprising:
 (a) providing a composition comprising a plurality of highly active achromosomal dynamic active systems (ADAS), wherein the ADAS have an initial ATP concentration of at least 1.25 mM and wherein the composition is substantially free of viable bacterial cells; and   (b) contacting the animal with an effective amount of the composition of step (a), thereby treating the animal.   
     
     
         121 . A method of treating an animal in need thereof, the method comprising:
 (a) providing a composition comprising a plurality of ADAS, wherein the ADAS are derived from parent bacteria having a reduction in the level or activity of a cell division topological specificity factor and wherein the composition is substantially free of viable bacterial cells; and   (b) contacting the animal with an effective amount of the composition of step (a), thereby treating the animal.   
     
     
         122 . The method of  claim 120  or  121 , wherein the animal has a cancer. 
     
     
         123 . The method of any one of  claims 120 - 122 , wherein the ADAS carries a chemotherapy cargo. 
     
     
         124 . The method of any one of  claims 120 - 122 , wherein the ADAS carries an immunotherapy cargo. 
     
     
         125 . A method of treating a plant in need thereof, the method comprising:
 (a) providing a composition comprising a plurality of highly active achromosomal dynamic active systems (ADAS), wherein the ADAS have an initial ATP concentration of at least 1.25 mM and wherein the composition is substantially free of viable bacterial cells; and   (b) contacting the plant or a pest thereof with an effective amount of the composition of step (a), thereby treating the plant.   
     
     
         126 . A method of treating a plant in need thereof, the method comprising:
 (a) providing a composition comprising a plurality of ADAS, wherein the ADAS are derived from parent bacteria having a reduction in the level or activity of a cell division topological specificity factor and wherein the composition is substantially free of viable bacterial cells; and   (b) contacting the plant or a pest thereof with an effective amount of the composition of step (a), thereby treating the plant.   
     
     
         127 . A composition comprising a plurality of ADAS, wherein the ADAS comprise an enzyme and wherein the enzyme alters a substrate to produce a target product. 
     
     
         128 . The composition of  claim 127 , wherein the substrate is present in the ADAS and wherein the target product is produced in the ADAS. 
     
     
         129 . The composition of  claim 127  or  128 , wherein the substrate is present in a target cell or environment to which the ADAS is delivered. 
     
     
         130 . The composition of any one of  claims 127 - 129 , wherein the enzyme is diadenylate cyclase A, the substrate is ATP, and the target product is cyclic-di-AMP.